Immunogenic peptides, and method of identifying same

Abstract

Immunogenic peptides, polynucleotides encoding immunogenic peptides, antibodies that selectively bind immunogenic peptides and methods of identifying immunogenic peptides are provided. The immunogenic peptides are representative of a structural element of a target protein. The methods of the invention are useful for identifying immunogenic peptides of a target protein having a known three dimensional structure, or of a target protein having a known amino acid sequence but an unknown three dimensional structure.

What is claimed is:  
     
         1 . A method for identifying an immunogenic peptide representative of a structural element of a target protein having a known three dimensional structure, comprising: 
 selecting epitopes of the target protein based on the three dimensional structure of the target protein, thereby obtaining selected epitopes;    detecting, in a molecular model of a polypeptide comprising a selected epitope linked to a scaffold protein, an epitope having a three dimensional conformation corresponding to the three dimensional structure of the epitope in the target protein, thereby identifying a candidate immunogenic peptide representative of a structural element of the target protein; and    detecting that antibodies induced by the candidate immunogenic peptide selectively bind to the target protein, thereby identifying an immunogenic peptide representative of a structural element of the target protein.    
     
     
         2 . The method of  claim 1 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         3 . The method of  claim 2 , wherein the viral coat protein comprises a Hepatitis B core protein.  
     
     
         4 . The method of  claim 1 , wherein the selected epitope is linked to the scaffold protein via a linker moiety.  
     
     
         5 . The method of  claim 4 , wherein the linker moiety comprises a peptide.  
     
     
         6 . The method of  claim 1 , wherein the polypeptide comprising a selected epitope linked to a scaffold protein is a fusion protein.  
     
     
         7 . The method of  claim 1 , wherein the scaffold protein comprises a poly(amino acid).  
     
     
         8 . The method of  claim 7 , wherein the poly(amino acid) comprises poly(lysine).  
     
     
         9 . The method of  claim 1 , wherein the target protein comprises a protein of an infectious microorganism.  
     
     
         10 . The method of  claim 9 , wherein the infectious microorganism is a bacterium.  
     
     
         11 . The method of  claim 10 , wherein the infectious microorganism causes anthrax.  
     
     
         12 . The method of  claim 11 , wherein the target protein comprises anthrax protective antigen.  
     
     
         13 . The method of  claim 11 , wherein the target protein comprises anthrax lethal factor.  
     
     
         14 . The method of  claim 2 , further comprising assembling a plurality of immunogenic peptides, each immunogenic peptide comprising an epitope linked to a viral coat protein, into a virus-like particle.  
     
     
         15 . The method of  claim 14 , wherein epitopes of the immunogenic peptides of the plurality are the same.  
     
     
         16 . The method of  claim 14 , wherein epitopes of the immunogenic peptides of the plurality comprise anthrax protective antigen epitopes and anthrax lethal factor epitopes.  
     
     
         17 . An immunogenic peptide identified according to the method of  claim 1 .  
     
     
         18 . An isolated peptide, consisting of: 
 amino acid residues 606 to 705 of SEQ ID NO:30;    amino acid residues 606 to 735 of SEQ ID NO:30;    amino acid residues 606 to 706 of SEQ ID NO:30;    amino acid residues 606 to 704 of SEQ ID NO:30;    amino acid residues 606 to 734 of SEQ ID NO:30;    amino acid residues 607 to 703 of SEQ ID NO:30;    amino acid residues 606 to 732 of SEQ ID NO:30;    amino acid residues 604 to 707 of SEQ ID NO:30;    amino acid residues 606 to 730 of SEQ ID NO:30; or    amino acid residues 606 to 733 of SEQ ID NO:30.    
     
     
         19 . An isolated antibody that selectively binds a peptide of  claim 18 , provided said antibody does not substantially bind a peptide comprising: 
 amino acid residues 596 to 735 of SEQ ID NO:30;    amino acid residues 679 to 693 of SEQ ID NO:30;    amino acid residues 703 to 722 of SEQ ID NO:30; or    amino acid residues 671 to 721 of SEQ ID NO:30.    
     
     
         20 . An isolated polynucleotide encoding the peptide of  claim 18 .  
     
     
         21 . An isolated peptide, consisting of: 
 amino acid residues 17 to 153 of SEQ ID NO:30;    amino acid residues 261 to 454 of SEQ ID NO:30; or    amino acid residues 487 to 594 of SEQ ID NO:30.    
     
     
         22 . A composition, comprising at least a first peptide of  claim 18  operatively linked to at least a first heterologous molecule.  
     
     
         23 . The composition of  claim 22 , wherein the first heterologous molecule comprises a linker moiety.  
     
     
         24 . The composition of  claim 23 , wherein the linker moiety comprises a peptide linker.  
     
     
         25 . The composition of  claim 24 , wherein the peptide linker comprises an oligo(glycine) linker.  
     
     
         26 . The composition of  claim 24 , wherein the peptide linker comprises a glycine-serine-alanine linker.  
     
     
         27 . The composition of  claim 22 , wherein the first heterologous molecule comprises a heterologous peptide.  
     
     
         28 . The composition of  claim 27 , which comprises a fusion protein.  
     
     
         29 . The composition of  claim 27 , wherein the heterologous peptide comprises a scaffold protein.  
     
     
         30 . The composition of  claim 29 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         31 . The composition of  claim 30 , wherein the viral coat protein comprises a Hepatitis B core protein.  
     
     
         32 . The composition of  claim 31 , wherein the Hepatitis B core protein consists of amino acid residues 1 to 77 and amino acid residues 80 to 149 of SEQ ID NO:34, and wherein the peptide of claim B 1 is operatively linked to amino acid residue 77 and to amino acid residue 80 of SEQ ID NO:34.  
     
     
         33 . The composition of  claim 32 , wherein the peptide of  claim 18  is operatively linked to amino acid residue 77 via a first linker moiety and is operatively linked to amino acid residue 80 via a second linker moiety.  
     
     
         34 . The method of  claim 33 , wherein the first linker moiety and the second linker moiety are the same.  
     
     
         35 . The method of  claim 33 , wherein each of the first linker moiety and the second linker moiety is a peptide.  
     
     
         36 . The composition of  claim 22 , further comprising at least a second heterologous molecule.  
     
     
         37 . The composition of  claim 36 , wherein at least the first heterologous molecule comprises a linker moiety or a scaffold protein.  
     
     
         38 . The composition of  claim 37 , wherein the first heterologous molecule comprises a linker moiety and the second heterologous molecule comprises a scaffold protein, and wherein the peptide of  claim 18  is operatively linked to the scaffold protein via the linker moiety.  
     
     
         39 . The composition of  claim 36 , comprising, in operative linkage, a first portion of a scaffold protein, a first peptide linker, the peptide of  claim 18 , a second peptide linker, and a second portion of the scaffold protein.  
     
     
         40 . The composition of  claim 39 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         41 . The composition of  claim 39 , which comprises an amino acid sequence as set forth in SEQ ID NO:36.  
     
     
         42 . The composition of  claim 40 , which comprises a plurality of polypeptides, wherein polypeptides of the plurality comprise a peptide of claim B 1 operatively linked to a viral coat protein.  
     
     
         43 . The composition of  claim 42 , wherein polypeptides of the plurality are assembled to form a virus-like particle.  
     
     
         44 . The composition of  claim 22 , wherein the first heterologous molecule comprises a tag.  
     
     
         45 . The composition of  claim 44 , wherein the tag comprises a peptide tag.  
     
     
         46 . The composition of  claim 45 , wherein the peptide tag comprises an oligo(histidine) tag.  
     
     
         47 . The composition of  claim 22 , which is comprises immunogenic peptide representative of a structural element of the target protein.  
     
     
         48 . The composition of  claim 47 , further comprising a carrier.  
     
     
         49 . The composition of  claim 48 , wherein carrier comprises an adjuvant.  
     
     
         50 . A polynucleotide encoding the composition of  claim 22 .  
     
     
         51 . The polynucleotide of  claim 50 , which comprises SEQ ID NO:35.  
     
     
         52 . The polynucleotide of  claim 50 , which is contained in a vector.  
     
     
         53 . The polynucleotide of  claim 52 , wherein the vector is a viral vector.  
     
     
         54 . The polynucleotide of  claim 50 , which is contained in a matrix.  
     
     
         55 . The polynucleotide of  claim 54 , wherein the matrix comprises liposomes or microbubbles.  
     
     
         56 . A method of stimulating an immune response in a subject, comprising administering the composition of  claim 22 , or a polynucleotide encoding the composition, to a subject under conditions suitable for stimulating an immune response.  
     
     
         57 . The method of  claim 56 , wherein the immune response comprises a protective immune response.  
     
     
         58 . The method of  claim 56 , wherein the subject is a mammalian subject.  
     
     
         59 . The method of  claim 56 , wherein the subject is a human subject.  
     
     
         60 . A method for identifying an immunogenic peptide representative of a structural element of a target protein having a known amino acid sequence but an unknown three dimensional structure, comprising: 
 generating a molecular model of a three dimensional structure of the target protein based on the three dimensional structure of a homologous protein;    selecting epitopes of the target protein based on the molecular model of the three dimensional structure of the target protein, thereby obtaining selected epitopes;    detecting, in a molecular model of a chimeric polypeptide comprising at least one selected epitope having a constrained structure, an epitope having a three dimensional conformation corresponding to the molecular model of the three dimensional structure of the epitope in the target protein, thereby identifying a candidate immunogenic peptide representative of a structural element of the target protein; and    detecting that antibodies induced by the candidate immunogenic peptide selectively bind to the target protein, thereby identifying an immunogenic peptide representative of a structural element of the target protein.    
     
     
         61 . The method of  claim 60 , wherein the chimeric polypeptide comprises a plurality of linked selected epitopes.  
     
     
         62 . The method of  claim 61 , wherein the selected epitopes of the plurality are the same.  
     
     
         63 . The method of  claim 60 , wherein the chimeric polypeptide further comprises at least one linker peptide, which is linked to at least one selected epitope of the chimeric polypeptide.  
     
     
         64 . The method of  claim 60 , wherein the constrained structure of the selected epitope is due to at least one disulfide bond in the chimeric polypeptide.  
     
     
         65 . The method of  claim 60 , wherein the constrained structure of the selected epitope is due to cyclization of the chimeric polypeptide.  
     
     
         66 . The method of  claim 60 , wherein the chimeric polypeptide further comprises a scaffold protein.  
     
     
         67 . The method of  claim 66 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         68 . The method of  claim 67 , wherein the viral coat protein comprises a Hepatitis B core protein.  
     
     
         69 . The method of  claim 66 , wherein the scaffold protein comprises a poly(amino acid).  
     
     
         70 . The method of  claim 69 , wherein the poly(amino acid) comprises poly(lysine).  
     
     
         71 . The method of  claim 66 , wherein the selected epitope is linked to the scaffold protein via a linker moiety.  
     
     
         72 . The method of  claim 71 , wherein the linker moiety comprises a peptide.  
     
     
         73 . The method of  claim 60 , wherein the target protein comprises a protein of an infectious microorganism.  
     
     
         74 . The method of  claim 73 , wherein the infectious microorganism is a bacterium.  
     
     
         75 . The method of  claim 74 , wherein the target protein comprises an autotransporter.  
     
     
         76 . The method of  claim 75 , wherein the autotransporter comprises an autotransporter as set forth in Table 1.  
     
     
         77 . The method of  claim 74 , wherein the infectious microorganism is a Shigella species.  
     
     
         78 . The method of  claim 77 , wherein the Shigella species is  Shigella flexneri.    
     
     
         79 . The method of  claim 77 , wherein the target protein comprises a Shigella protease involved in colonization (pic) gene product.  
     
     
         80 . The method of  claim 74 , wherein the infectious microorganism is a Yersinia species.  
     
     
         81 . The method of  claim 80 , wherein the Yersinia species is  Yersinia pestis.    
     
     
         82 . The method of  claim 80 , wherein the target protein comprises a Yersinia murine toxin (YMt).  
     
     
         83 . The method of  claim 67 , further comprising assembling a plurality of immunogenic peptides, each immunogenic peptide comprising an epitope linked to a viral coat protein, into a virus-like particle.  
     
     
         84 . The method of  claim 83 , wherein epitopes of the immunogenic peptides of the plurality are the same.  
     
     
         85 . An immunogenic peptide identified according to the method of  claim 60 .  
     
     
         86 . An isolated peptide, consisting of a peptide as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1 1, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.  
     
     
         87 . An isolated polynucleotide encoding the peptide of  claim 86 .  
     
     
         88 . The polynucleotide of  claim 87 , which is contained in a vector.  
     
     
         89 . The polynucleotide of  claim 88 , wherein the vector is a viral vector.  
     
     
         90 . The polynucleotide of  claim 87 , which is contained in a matrix.  
     
     
         91 . The polynucleotide of  claim 90 , wherein the matrix comprises liposomes or microbubbles.  
     
     
         92 . A composition, comprising at least a first peptide of  claim 86  operatively linked to at least a first heterologous molecule.  
     
     
         93 . The composition of  claim 92 , comprising a plurality of peptides of  claim 86 , wherein the peptides of the plurality are linked to each other.  
     
     
         94 . The composition of  claim 93 , wherein peptides of the plurality are the same.  
     
     
         95 . The composition of  claim 92 , wherein the first heterologous molecule comprises a linker moiety.  
     
     
         96 . The composition of  claim 95 , wherein the linker moiety comprises a peptide linker.  
     
     
         97 . The composition of  claim 92 , wherein the first heterologous molecule comprises a first heterologous peptide.  
     
     
         98 . The composition of  claim 97 , wherein the first heterologous peptide comprises a scaffold protein.  
     
     
         99 . The composition of  claim 98 , wherein the scaffold protein comprises a poly(amino acid).  
     
     
         100 . The composition of  claim 99 , wherein the poly(amino acid) comprises poly(lysine).  
     
     
         101 . The composition of  claim 98 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         102 . The composition of  claim 101 , wherein the viral coat protein comprises a Hepatitis B core protein.  
     
     
         103 . The composition of  claim 102 , wherein the Hepatitis B core protein consists of amino acid residues 1 to 77 and amino acid residues 80 to 149 of SEQ ID NO:34, and wherein the peptide of  claim 86  is operatively linked to amino acid residue 77 and to amino acid 80 of SEQ ID NO:34.  
     
     
         104 . The composition of  claim 101 , comprising a plurality of peptides of  claim 86 , wherein each peptide of the plurality is operatively linked to a viral coat protein.  
     
     
         105 . The composition of  claim 104 , which are assembled to form a virus-like particle.  
     
     
         106 . The composition of  claim 92 , further comprising at least a second heterologous molecule.  
     
     
         107 . The composition of  claim 106 , wherein at least the first heterologous molecule comprises a linker moiety or a scaffold protein.  
     
     
         108 . The composition of  claim 106 , wherein the first heterologous molecule comprises a linker moiety and the second heterologous molecule comprises a scaffold protein, and wherein the linker moiety operatively links the peptide of claim NI to the scaffold protein.  
     
     
         109 . The composition of  claim 106 , comprising, in operative linkage, a first portion of a scaffold protein, a first peptide linker, the peptide of claim N1, a second peptide linker, and a second portion of the scaffold protein.  
     
     
         110 . The composition of  claim 109 , wherein the scaffold protein comprises a viral coat protein.  
     
     
         111 . The composition of  claim 92 , wherein the first heterologous molecule comprises a tag.  
     
     
         112 . The composition of  claim 92 , wherein the first heterologous molecule comprises a carrier protein.  
     
     
         113 . The composition of  claim 92 , which is an immunogenic composition.  
     
     
         114 . The composition of  claim 113 , further comprising an adjuvant.  
     
     
         115 . A method of stimulating an immune response in a subject, comprising administering the composition of  claim 92 , or a polynucleotide encoding the composition, to a subject under conditions suitable for stimulating an immune response.  
     
     
         116 . The method of  claim 115 , wherein the immune response comprises a protective immune response.  
     
     
         117 . The method of  claim 115 , wherein the subject is a mammalian subject.  
     
     
         118 . The method of  claim 115 , wherein the subject is a human subject.